Abstract

Purpose: The interplay between genetic susceptibility and exposure to carcinogens has been shown to be involved in the etiology of many solid tumors. We studied the frequency and clinical correlates of polymorphisms resulting in deletions of two genes involved in the detoxification of potentially carcinogenic agents, glutathione S-transferase (GST)-M1 and GSTT1 in patients with Hodgkin’s lymphoma (HL).

Experimental Design: The prevalence of gene deletions in 90 patients with HL was compared with a case-matched cohort of 176 normal blood donors. GST gene polymorphisms were studied using a multiplex PCR method, including the BCL2 gene as an internal control.

Conclusions: The GSTT1-null genotype may increase the risk for HL and is associated with favorable prognostic factors, and the presence of at least one GST deletion indicates an improved disease-free survival.

INTRODUCTION

HL3
is a heterogeneous malignancy, and little is known about the etiology of this disease. Epidemiological data support the idea that different pathogenetic factors may play a role in different subsets of the disease. Infection with the EBV and an abnormal immune reaction to EBV have been identified as a pathogenetic factor in at least a subset of pts (1)
. The association between EBV and HL is particularly evident in HL cases occurring in developing countries (2)
. Increased ORs for a positive EBV tumor status have been found in the mixed cellularity versus the nodular sclerosis histotype, children versus young adults, males versus females, and Hispanics versus Caucasians (3)
. Other possible risk factors for HL, which have been identified by epidemiological studies, are genetic susceptibility, environmental exposure to herbicides, and certain occupations (4, 5, 6, 7, 8, 9, 10)
. A genetic predisposition is supported by the finding of a familial clustering of HL cases (4, 8, 9, 10)
. Familial HL is characterized by only one major incidence peak between ages 15 and 34 years, in contrast to the broader incidence of sporadic HL, and EBV appears not to play an important role in familial HL (10)
. A specific genetic alteration has not been identified, and the familial clustering could reflect a common environmental exposure (4, 9)
. Some studies have suggested that the incidence of HL in young adults, in particular among women, is rising since the 1970s, and this could reflect changes in carcinogen exposure in the different generations (11)
. Taken together, these data suggest that a genetic susceptibility to the tumorigenic effects of environmental agents could be involved in the pathogenesis of HL.

Environmental carcinogens are metabolized in vivo by enzymatic reactions that are classically divided into two categories: (a) the Phase I enzymes, mediating oxidation and activation, which are almost exclusively cytochrome P450 enzymes; and (b) the Phase II enzymes, mediating glucorinidation, acetylation, or conjugation with glutathione-S and most often creating a more water-soluble conjugate, which may be less toxic and more readily excretable (12)
. Polymorphisms in several genes of these enzymatic pathways are believed to be key factors in determining cancer susceptibility to toxic or environmental chemicals (13)
. GSTs are involved in the conjugation of several environmental pollutants as polyaromatic hydrocarbons but also of anticancer drugs, including alkylating agents, anthracyclines, and cyclophosphamide (14, 15, 16)
. Conjugation of substrates is not necessarily beneficial, because conjugation of monohalomethanes and ethylene oxide by the GST θ class enzymes is detoxifying, whereas conjugation of the chemical dichloromethane or brominated tri-halomethanes by the same enzyme yields a mutagenic metabolite (16)
. Moreover, also cytoprotective agents, such as phenethylisothiocyanate, a strong inducer of Phase II detoxifying enzymes, are substrates of GST enzymes (17)
. Thus, enzymatic deficiencies of GST enzymes have complex metabolic consequences, and the kind of exposure is suspected to be important whether the GST genotype confers a decreased or increased risk of cancer. Polymorphic deletions in two GST genes, GSTM1 and GSTT1, are present in a large proportion of the population (15, 16)
. Homozygous deletions cause loss of enzymatic function and have been shown to be important risk factors for solid tumors (13)
. More recently, the role of GST genotypes in the pathogenesis of hematological neoplasms, in particular of acute leukemias, has been addressed (18, 19, 20, 21)
. There are only few reports thus far on the association of GST genotypes and lymphoma risk (22, 23, 24)
. A recent review discussed the potential role for GST polymorphisms in the etiology of treatment-related complications in Hodgkin’s disease (25)
.

In this study, we examined the frequency of GSTM1 and GSTT1 deletions in pts with HL in comparison with a control cohort, correlated the genotype to patient characteristics, and determined the prognostic value of the genotypic status.

PATIENTS AND METHODS

Patient Characteristics.

Our retrospective analysis included 90 pts (median age 33 years, range 14–71 years; 36 females and 54 males), diagnosed with HL between April 1977 and July 2002. Patient characteristics are detailed in Table 1⇓
. Peripheral blood samples were obtained at the time of initial diagnosis or during follow-up. All but 1 patient were treated with standard chemotherapy regimens: 47 pts received ABVD, 29 pts a modified Stanford V regimen (substituting 6 mg/m2 mechlorethamine with 650 mg/m2 cyclophosphamide), 7 pts an ABVD/MOPP hybrid regimen, 5 pts BEACOPP, and 1 pt VEBEP (26, 27, 28)
. In 42 pts, radiotherapy was included for consolidation. In 73 pts, a complete response was achieved after first-line treatment.

The peripheral blood of 176 healthy individuals, matched for sex and age (69 females, 107 males; median age 38 years, range 19–71 years), was used as control. Controls had a medical history negative for any type of cancer and were not related to the pts. All pts and controls were Italians. Informed consent was obtained from all pts, according to institutional guidelines.

DNA Extraction and Amplification.

Leukocytes were freshly isolated from the peripheral blood of pts and controls after erythrocyte hypotonic lysis. Cells were then washed two times with PBS at 400 grams and 4°C for 7 min, and DNA was extracted using DNAzol (Invitrogen, Carlsbad, CA), following the manufacturer’s instructions.

Homozygous deletions of GSTM1 and GSTT1 were studied using a multiplex PCR technique, including primers for the housekeeping gene BCL-2 as internal control (29)
. PCR was carried out in a 50-μl mixture containing 100 ng of genomic DNA and a “master” mix of PCR buffer, 2.5 mm MgCl2, 250 nm deoxynucleotide triphosphates, 1.25 units of Taq polymerase (Taq Platinum; Invitrogen), and 0.8 μm the following primers: (a) 5′-TTCCTTACTGGTCCTCACATCTC, 5′-TCACCGGATCATGGCCAGCA for GSTT1; (b) 5′-GAACTCCCTGAAAAGCTAAAGC, 5′-GTTGGGCTCAAATATACGGTGG for GSTM1; and (c) 5′-GCAATTCCGCATTTAATTCATGG-3′, 5′-GAAACAGGCCACGTAAAGCAAC-3′ for BCL-2. Amplification consisted of 35 cycles of denaturation at 94°C for 1 min, annealing at 62°C for 1 min, and extension at 72°C for 1 min. This results in a fragment of 480 bp for GSTT1, 219 bp for GSTM1, and 154 bp for BCL-2. PCR products were evaluated on an ethidium bromide-stained 2% agarose gel. A positive and negative control, containing water instead of DNA, was included in all PCR reactions.

Statistical Analysis.

The statistical significance of the differences between HL cases and controls was calculated using Fisher’s exact test (two sided). Absolute ORs were determined separately for GSTM1 and GSTT1 deletions and are given within 95% CIs. Age and gender were included as covariables. The age of 45 years was used as a cutoff value because it represents an important prognostic cutoff point, and it reflects also the cutoff of the bimodal age distribution of HL (30, 31)
. In addition, Fisher’s exact test was used to examine for associations of GST genotypes with patient characteristics of established prognostic significance as histotype, stage, presence of B symptoms, and alterations of laboratory parameters (30, 31)
. Survival curves were estimated using the Kaplan-Meier product limit method. Differences in the survival curves were evaluated with the Log-rank test. Disease-free survival was defined as the time to relapse for pts achieving complete remission. Cox regression was used for multivariate models that analyzed differences between groups adjusting for patient characteristics that had prognostic significance in the univariate analysis. A two-sample test of equality of proportions was used to calculate the sample size of patient and control cohorts to verify our findings of associations between GST genotypes and disease risk and patient characteristics. All computations were performed using the Stata 6.0 software (Stata Corp., College Station, TX).

RESULTS

Frequency of GST Deletions.

We studied 90 pts with HL and 176 normal blood donors matched for age and sex. The frequency of GST deletions in the control population was as expected for Caucasians: (a) GSTM1 null, 49.4%; (b) GSTT1 null, 17.6%; and (c) GSTT1/M1 double null, 8% (Table 2)⇓
. We found a higher prevalence of the GSTT1 deletions among pts with HL (28.9%, P = 0.04). This translated into a significant increased risk of HL for individuals with the GSTT1-null genotype (OR, 1.9; 95% CI, 1.04–3.46). The overall frequency of GSTM1 deletions and double null genotype did not differ between HL pts and controls (42.2 versus 49.4%, P = 0.3, and 8.9 versus 8%, P = 0.7, respectively). We next analyzed whether the risk of HL could be increased in a subgroup of pts, grouped according to age and sex (Table 3)⇓
. An increased frequency of GSTT1 deletions was observed in pts younger than 45 years (30 versus 16.8%, P = 0.02), resulting in an OR of 2.5 (95% CI, 1.2–5). This risk was even higher in females aged <45 years (OR, 6.1; 95% CI, 1.6–23, P = 0.008). Considering in the same way age and sex as covariables, no difference in HL risk was observed for individuals with GSTM1 deletions.

GST Genotype and Prognosis.

As GST enzymes are also involved in the metabolism of cytotoxic agents, we next asked whether GST genotypes were predictive for response to therapy and prognosis. Forty-seven pts were treated with ABVD, 29 pts with a modified Stanford V regimen, and 15 pts received other regimens (BEACOPP, MOPP/ABVD, and VEBEP). In 42 pts, radiotherapy was included for consolidation. In 73 pts, a complete response was achieved after first-line treatment. There were no differences in the remission rates nor event-free and overall survival when grouping pts according to the different therapy protocols (data not shown). The remission rates to first-line therapy did not differ according to GST genotype. Pts with at least one GST deletion (GSTM1− or GSTT1−) had a significantly better disease-free survival when compared with those with undeleted GST genes (GSTM1+/GSTT1+; P = 0.012; Fig. 1⇓
). Overall survival was not influenced by the GST genotypes. We next analyzed whether the presence of at least one GST-null genotype was an independent prognostic factor. We first analyzed the prognostic value of established prognostic factors, as age, sex, stage of the disease, presence of bulky disease or B-symptoms, and abnormal laboratory parameters in a univariate analysis (Table 5)⇓
. Stage IV and Hb levels < 12 grams/dl proved to be of prognostic value in our patient group. We therefore included these parameters in a multivariate analysis using the Cox regression model (Table 5)⇓
. The presence of at least one GST deletion continued to be of positive prognostic value.

DISCUSSION

In the effort to improve our understanding on the genetic predisposition in HL, we determined the frequency of deletions in the detoxification genes GSTM1 and GSTT1 in comparison with a matched control group, correlated the genotypic status to patient characteristics, and studied its prognostic impact. The frequency of the GSTT1-null genotype was significantly increased in pts with HL compared with controls, conferring a 1.9-fold increase in the risk of HL. The risk for HL was particularly increased in young females, younger carriers of the GSTT1-null genotype. As the etiology of HL is probably as heterogeneous as the disease is, this association could indicate a pathogenetic role of an altered carcinogen metabolism in a distinct subset of the disease. In the same line, HL in female pts in the age between 15 and 45 years is characterized by the nodular sclerosing histotype and not associated with EBV, and the incidence is rising, suggesting changes in carcinogen exposure (3, 4, 11)
. These circumstances nicely fit to the idea that genetic susceptibility defined by the polymorphism in a detoxification gene predisposes to the development to HL in this group. Thus far, there are only few reports studying the association between GST genotypes and lymphoid malignancies. An increased frequency of GSTT1 genotype has been observed in pts with non-HL and in African-American children with acute lymphoblastic leukemia (19, 22)
. Sarmanova et al. (24)
found an increased prevalence of the Val/Val genotype of GSTP1-exon 5 in pts with HL, and this difference was particularly evident in females. Taken together, these findings argue for a role of xenobiotic metabolism defects in the pathogenesis of lymphoid malignancies, raising the possibility of common etiological mechanisms underlying some cases of these diseases.

The GSTT1-null genotype was associated with a limited stage of disease and low ESR, both well-established favorable prognostic factors. The finding of an association of the GSTT1-null genotype with favorable prognostic factors appears at first unexpected, given that this is a risk factor for HL. However, the GSTT1-null genotype appears to be associated with Hodgkin’s disease risk in particular in young females, a patient group characterized by favorable prognosis. Age > 45 years and male sex are two of seven independent prognostic factors elaborated by the “International Factors Project on Advanced Hodgkin’s Disease.” (31)
. We further analyzed whether the GSTT1-null genotype could identify a prognostic subgroup. To our knowledge, this is the first report on the association between GST genotype and clinical outcome in HL. The presence of at least one GST deletion proved to be a favorable prognostic factor. As the absence of GST enzymes could simply reflect a biologically distinct, less aggressive disease, we analyzed also for possible associations of the presence of at least one GST deletion with patient characteristics. We found no such associations (data not shown). Furthermore, in a multivariate analysis, the presence of at least one GST deletion continued to be of independent prognostic significance. We therefore assume that the impact of the GST genotype on disease-free survival stems from the role of these enzymes in the metabolism of chemotherapeutic drugs, such as alkylating agents and anthracyclines, which are used in the treatment regimens of HL pts. This detoxification can protect the cells from the injury of the cytotoxic chemotherapy. GST enzymes inactivate alkylating agents by catalyzing the direct conjugation of these substrates. Another mechanism of detoxification is the neutralization of reactive compounds, such as electrophiles, induced by the chemotherapeutic agent (14)
. An example for the latter is the detoxification of products of lipid peroxidation, which can be caused by anthracyclines. We hypothesize that the most likely link between a better prognosis and the GSTT1- and/or M1-null genotype would be the conjugation of one or more substrates that these two enzymes have in common. GST isozymes function on a wide range of substrates, mostly with a low binding activity. Transfection studies may be helpful to define the substrates involved in the anticancer drug resistance conferred by GST (32)
. Tumor cells expressing high levels of GST enzymes have been shown to be more resistant in vitro and in vivo to chemotherapy (33)
. In this line, Stanulla et al. (34)
showed a reduced risk of relapse in childhood B-cell acute lymphoblastic leukemia pts having GSTM1- or GSTT1-null genotypes. On the other hand, we had shown that the presence of at least one GST deletion was an independent prognostic factor in pts with acute myeloid leukemia associated with poor prognosis (29)
. AML pts with GSTM1 or GSTT1 deletion had also an increased resistance to chemotherapy reflecting probably a biologically more aggressive disease. In the same line, the presence of at least one GSTM1 allele was of significant beneficial effect on treatment outcome in pediatric pts with non-HL (23)
. Thus, the prognostic impact of deletions of GST enzymes appears to be different in diverse clinical situations.

In conclusion, GSTT1 deletions increased the risk for Hodgkin’s disease, particularly in women younger than 45 years. This genotype was associated with favorable prognostic factors as limited stage and lower ESR, and the presence of at least one GST deletion indicated an improved disease-free survival. However, larger studies, including more pts, are needed to better define the complex associations of host genetic variations with disease characteristics and outcome in lymphoid neoplasias. On the basis of our current findings, we estimated the sample size to confirm our finding of an association between GSTT1-null genotype and Hodgkin’s disease risk. Using the design of a 2:1 ratio between controls and pts, 173 pts and 346 controls have to be enrolled to confirm the difference with a power level of 80%, whereas a cohort of 229 pts will be needed for a power level of 90%. As well, 231 pts will be needed to confirm the associations between patient characteristics and GST polymorphisms at a 90% power level.

Footnotes

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